Optimized chemical probes for REV-ERBα

Article abstract of DOI:10.1021/jm400458q

REV-ERBα has emerged as an important target for regulation of circadian rhythm and its associated physiology. Herein, we report on the optimization of a series of REV-ERBα agonists based on GSK4112 (1) for potency, selectivity, and bioavailability.(1) Potent REV-ERBα agonists 4, 10, 16, and 23 are detailed for their ability to suppress BMAL and IL-6 expression from human cells while also demonstrating excellent selectivity over LXRα. Amine 4 demonstrated in vivo bioavailability after either iv or oral dosing.

Full text of DOI:10.1021/jm400458q

Article
pubs.acs.org/jmc
Optimized Chemical Probes for REV-ERBα
Ryan P. Trump,*,† Stefano Bresciani,‡ Anthony W. J. Cooper,§ James P. Tellam,‡ Justyna Wojno,‡
John Blaikley,∥ Lisa A. Orband-Miller,† Jennifer A. Kashatus,† Mohamed Boudjelal,§,# Helen C. Dawson,†
Andrew Loudon,⊥ David Ray,∥ Daniel Grant,† Stuart N. Farrow,§ Timothy M. Willson,†
and Nicholas C. O. Tomkinson‡
†Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina 27709-3398, United States
‡WestCHEM, Department of Pure and Applied Chemistry, Thomas Graham Building, University of Strathclyde, Glasgow, G1 1XL,
U.K.
§Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Hertfordshire, SG1 2NY, U.K.
∥Faculty of Medical and Human Sciences, University of Manchester, Manchester, M13 9PT, U.K.
⊥Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, U.K.
S
* Supporting Information
ABSTRACT: REV-ERBα has emerged as an important target for regulation of circadian rhythm and its associated physiology.
Herein, we report on the optimization of a series of REV-ERBα agonists based on GSK4112 (1) for potency, selectivity, and
bioavailability.1 Potent REV-ERBα agonists 4, 10, 16, and 23 are detailed for their ability to suppress BMAL and IL-6 expression
from human cells while also demonstrating excellent selectivity over LXRα. Amine 4 demonstrated in vivo bioavailability after
either iv or oral dosing.
INTRODUCTION
■
The circadian clock aligns all the tissues in most organisms with
the day and night cycle of our planet. Through a transcriptional
mechanism, the clock controls many important biological
pathways, such as metabolism, inflammation, and sleep−wake
cycles.2 REV-ERBα is a nuclear receptor that has been
demonstrated, upon activation by heme, to form a complex
with cofactors that represses the transcription of target genes.3
REV-ERBα is at the heart of the circadian clock and is a
Figure 1. Reported REV-ERBα agonists 1−3.
mechanism by which the circadian clock gates inflammatory
response and controls the metabolic state of the organism.4
the nuclear receptor LXRα, a potential liability for interpreta-
tion of results from cell-based and animal pharmacology.6 Lack
of selectivity for any chemical probe introduces challenges.7
Specifically, LXRα has been shown to have effects in anti-
inflammatory and metabolic pathways that overlap with those
under investigation for the REV-ERBα. Herein we describe our
optimization of the tertiary amine series of REV-ERBα agonists
to address these liabilities that allow further exploration of the
role of REV-ERBα in circadian control and inflammation.
Investigation into the function of REV-ERBα will provide
further insights into circadian biology and may identify the
receptor as a therapeutic target for a variety of diseases.
Pharmacological investigations into the biological role of the
REV-ERBα have used the suboptimal chemical probe GSK4112
(1) and its analogs SR9011 (2) and SR9009 (3) (Figure 1).1,5
All three compounds are closely related by a common tertiary
amine core and two of three identical substituents of the amine.
Use of these compounds to interrogate REV-ERBα biology is
complicated by high metabolic clearance rates that necessitate
high dosing to achieve meaningful levels of exposure in vivo. In
addition, the tertiary amine chemotype has known activity on
Received: March 29, 2013
© XXXX American Chemical Society
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Table 1. REV-ERBα and LXRα Activity of Amine 1 Analogues
LXRα
BMAL1
REV-ERBα
EC50
IC50
fold
supp
(%)
phase
delay
a
b
c
compd
R1
R2
R3
cLogP
(μM) selectivity
1
tBuO2C-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Me-C6H4-
4-F-C6H4-
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrothiophen-2-yl
5-nitrilethiophen-2-yl
4-Br-5-Me-thiophen-2-yl
benzo[b]thiophen-2-yl
3-(1H-pyrrol-1-yl)thiophen-2-yl
thiazol-2-yl
5.2
5.3
4.8
6.8
5.6
5.7
5.9
4.1
3.9
3.5
3.8
3.9
3.3
3.3
4.3
3.8
5.7
5.7
5.5
3.0
2.5
3.2
3.3
3.0
3.4
3.3
0.50
>50
>50
0.050
0.10
0.063
0.16
0.16
0.16
0.16
0.20
0.20
0.20
0.40
0.40
0.20
0.25
0.40
0.63
>50
>50
0.20
0.20
0.63
0.63
>50
5.0
13
10
6
0
2
1-N-pentylcarboxamidepyrrolidin-3-yl
1-ethylcarboxylate-pyrrolidin-3-yl
4-Cl-2-Me-C6H3-
Ph-
14
1.3
1.3
0
3
6.3
63
20
4
1259
>100
320
100
79
21
5
>100
20
NT
38
NT
0
6
2-F-C6H4-
7
isobutyl-
16
21
0
8
3-pyridyl-
13
NT
23
NT
0
9
3-pyridyl-
40
250
250
160
130
160
25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
3-pyridyl-
40
14
0
3-pyridyl-
2-Me-C6H4-
3-Me-C6H4-
4-OMe-C6H4-
3-OMe-C6H4-
3-CF3-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-F-C6H4-
32
37
0
3-pyridyl-
25
41
0.8
NT
0.5
NT
0
3-pyridyl-
32
NT
42
3-pyridyl-
10
3-pyridyl-
16
40
NT
23
3-pyridyl-
20
100
130
100
63
3-pyridyl-
32
16
0
3-pyridyl-
40
24
0
3-pyridyl-
40
30
0
3-pyridyl-
50
<1
NT
NT
NT
37
NT
NT
NT
0
3-pyridyl-
1H-imidazol-2-yl
>100
NT
16
NT
NT
79
3-pyridyl-
5-nitrilethiophen-2-yl
5-nitrilethiophen-2-yl
5-nitrilethiophen-2-yl
5-nitrilethiophen-2-yl
5-nitrilethiophen-2-yl
3-pyridyl-
3,4-F2-C6H3-
4-OMe-C6H4-
2,5-F2-C6H3-
2-F-C6H4-
3-pyridyl-
>100
6.3
32
>160
10
NT
27
NT
0
3-pyridyl-
3-pyridyl-
<1
6
0
a
b
EC50 values are in μM. All active compounds demonstrated a 30−60% increase in NCOR peptide recruitment. Suppression of BMAL1 expression
c
after 40 h of 20 μM compound treatment. Delay of peak of second cycle in hours of 20 μM treated cells relative to DMSO treated cells.
PGC1-β. Full curve analysis of compounds 1−3 demonstrated
the dose-dependent recruitment of PGC1-β peptide to REV-
ERBα (Figure 2). This result demonstrated that amines 2 and 3
do bind to the REV-ERBα protein, as reported, to induce a
RESULTS AND DISCUSSION
■
To deliver a tool suitable for in vivo studies, we evaluated the
literature compounds 1−3 to assess opportunities for improve-
ment. First we investigated the ability of the compounds 1−3
to bind to both REV-ERBα and LXRα. The tertiary amine 1
induced recruitment of cofactor NCOR peptide fragment to
purified REV-ERBα protein. This result indicated that the
compound binds to and induces a change, potentially
conformational, in the protein that alters the peptide
recruitment profile (Table 1). Interestingly, neither compound
2 or 3 increased recruitment of the NCOR peptide fragment to
REV-ERBα in vitro, a result that does not preclude the binding
of compound 2 or 3 to the REV-ERBα but only that the
potential binding of the compounds does not induce the same
change in the protein to allow recruitment of our NCOR
peptide fragment to our purified REV-ERBα. Amines 2 and 3
were reported to bind to the protein and have other activities
associated with REV-ERBα activation, so we investigated the
compounds’ ability to activate the receptor by a different
mechanism.5 To determine if these two compounds were
recruiting a different set of peptides to the REV-ERBα protein,
we performed a scan of cofactor derived peptide fragments
recruited to REV-ERBα in the presence of amine 1 (see
Supporting Information Figure 1). Aside from NCOR and
SMRT, the only other tested peptide fragments that showed
significant interaction with the REV-ERBα was a fragment of
Figure 2. Effect of compounds 1−3 on PGC1-β peptide fragment
recruitment to REV-ERBα.
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change in the protein that is different from that produced by
amine 1. These data raise the intriguing possibility that the
different peptide recruitment profiles of the compounds may
result in distinct biological activities. They also indicate that
PGC1-β may have a yet uninvestigated role in REV-ERBα
pharmacology. The exploration of these possibilities is currently
under investigation and is beyond the scope of this paper.
Each of the compounds 1−3 was able to displace a
radioligand from the LXRα binding site, confirming that the
compounds bind to the LXRα at ∼10 μM (Table 1). We
investigated if the observed binding of the compounds to both
REV-ERBα and LXRα induced related effects in a living cell.
To investigate REV-ERBα cellular efficacy, we measured the
ability of agonists 1 and 2 to inhibit IL-6 production from
human THP-1 cells following LPS stimulation (Figure 3a). To
cells. Since activation of LXRα has been reported to have
diverse effects on multiple pharmacological pathways, including
inflammation, cholesterol metabolism, and insulin resistance,
we set a key goal for this project to improve the selectivity
profile for the series.6
In addition to the observed off-target activity, compounds 1−
3 have relatively high cLogP values of >5 that may contribute to
off-target binding, particularly with nuclear receptors, and the
high clearance observed in reported mouse DMPK studies.8
The goal of our synthetic efforts was therefore to provide an
effective and readily accessible in vivo probe for REV-ERBα
that could be used as an efficient tool to explore the biological
profile of this fascinating receptor. To improve on the
significant shortfalls of 1−3, it was crucial to increase selectivity
over LXRα to allow clear and precise interpretations of
subsequent data and secondarily to reduce the lipophilicity of
the compounds. Preparation of the target tertiary amines was
through a series of reductive amination procedures (Scheme 1).
Scheme 1. Synthesis of Tertiary Amine Compounds
The overall process could be carried out under ambient
reaction conditions or accelerated through microwave irradi-
ation. It also proved possible to carry out the entire reaction
sequence through a one-pot two-step protocol allowing rapid
access to a range of tertiary amine scaffolds for evaluation.
Pertinent data in this ligand development on a selection of the
amines prepared are collected in Table 1.
Figure 3. Effects of compounds 1 and 2 on cellular makers of (a)
REV-ERBα and (b) LXRα activity. THP-1 cells were stimulated with
LPS, 10 μg/mL, in the presence of DMSO, 1 (10 μM), or compound
2 (10 μM). After 6 h the cells were lysed and the mRNA was analyzed
using RT-qPCR. Both compounds significantly repressed IL-6 mRNA
expression but increased ABCA1 mRNA expression (∗∗, p < 0.01, t
test). A representative experiment of three experiments is shown.
Initial efforts focused on replacing the tert-butyl ester arm
(R1) of compound 1. Alternative substitution at this position
provided multiple options for improved REV-ERBα activity and
LXRα selectivity but often at the cost of an increase in
compound cLogP (Table 1); however, significant improve-
ments in the overall compound profile proved to be possible.
Replacement of the tert-butyl ester with both substituted benzyl
(4−6) and alkyl (7) groups was not only well tolerated but led
to stunning improvements in LXRα selectivity. Although many
of these changes resulted in an increase in cLogP, it showed
that LXRα activity could ultimately be removed from this
tertiary amine series through simple structural modification. In
order to address the lipophilicity issue, a variety of heterocyclic
substituents were examined in this position, the vast majority of
which were not tolerated (data not shown); however,
introduction of a 3-pyridyl group (e.g., 8) provided a modest
increase in REV-ERBα activity (EC50 = 0.16), significant
improvement in LXRα selectivity (79 fold), and importantly a
meaningful lowering in cLogP (4.1).
Using compound 8 as the basis for continued exploration of
scaffold modification, we examined variation of the 4-
chlorobenzyl arm (R2) of compound 1. In contrast to
substitution of R1, both alkyl chains and heterocyclic
substituents were not tolerated in this position with all changes
resulting in compounds with no significant activity at REV-
ERBα (data not shown). It was possible to replace this
substituent with a variety of substituted benzyl groups (e.g., 9−
15) that provided multiple options that maintained REV-ERBα
activity and concurrently improved LXRα selectivity and
assess the cellular activity of the compounds in an LXRα-
dependent system, we compared the effects of the compounds
1 and 2 on expression of ABCA1 in THP-1 cells (Figure 3b).
Both compounds were able to significantly inhibit LPS-
stimulated IL-6 mRNA expression and up-regulate ABCA1
mRNA levels, indicating significant cellular activity of each
compound on both REV-ERBα and LXRα dependent
phenotypes that correlated with the ability of the compounds
to bind to target proteins.
The original report on compound 2 described a lack of
activity in a chimeric LXRα-Gal4 reporter gene assay in which
the binding domain of LXRα was fused to a yeast transcription
factor. However, our results indicated that the compounds
bound directly to purified LXRα protein and induced cellular
affects through activation of the endogenous LXRα which
contains multiple domains important for signal transduction
that are absent in the chimeric LXRα-Gal4 construct. While we
were able to replicate the published results with an artificial
LXRα-Gal4 reporter assay (data not shown),5 the assay may
not be an appropriate system to measure the selectivity of the
current series of REV-ERBα agonists because of the artificial
nature of the LXRα employed. Another possibility is that, as
noted above, compounds 2 and 3 induced a different profile of
peptide recruitment to the REV-ERBα and may be acting as
modulators of NR action, achieving selective activation of
different NR responses. Importantly, compounds 1−3 bind to
LXRα and up-regulate a marker of LXRα activation in primary
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reduced cLogP. For example, introduction of a 4-fluorobenzyl
unit (10) lowered the cLogP significantly (3.5) and improved
selectivity over LXRα to >250-fold. Other changes at R2 such as
the introduction of a 4-methoxybenzyl substituent (13)
provided a similar and substantial improvement in overall
compound profile.
suppression and shift of the BMAL oscillation curve in a dose-
dependent manner.
During the evolution of this SAR knowledge, compounds 4
(GSK2945), 10 (GSK0999), 16 (GSK5072), and 23
(GSK2667) (Figure 5) were selected for further profiling and
evaluation of their suitability to be adopted as in vivo probes for
REV-ERBα.
In parallel to these modifications, an array of compounds was
prepared replacing the nitro- substituent on the thiophene ring
(R3). Interestingly, the majority of replacements for the
nitrothiophene ring system with alternative polar five-
membered ring heterocycles as exemplified by compounds 20
and 21 resulted in complete loss of activity in the primary
NCOR peptide recruitment assay. It did prove to be possible to
introduce different substituents to the thiophene ring and
maintain REV-ERBα potency and improve LXRα selectivity as
exemplified by compounds 17 and 18; however, these changes
often proved to be detrimental to the cLogP. One crucial
exception to this trend was introduction of the 5-nitrile
thiophene in compound 16, which not only maintained REV-
ERBα selectivity (EC50 = 0.2) but also resulted in a significant
drop in cLogP (3.8) and a slight improvement in LXRα
selectivity (100-fold).
Combination of the optimal substituents discovered for R1,
R2, and R3 resulted in the tertiary amines 22−26 which on
evaluation revealed the key compound 23 which maintained
potency in the NCOR peptide recruitment assay and also
provided the desired LXRα selectivity profile and reduced
lipophilicity.
Figure 5. Key compounds selected during study for further evaluation
as potential probes.
Initially, to confirm the ability of the compounds to induce
REV-ERBα-driven phenotypes in a cellular system, the ability
to affect the circadian expression of BMAL in synchronized
U2OS cells was examined. By stably expressing a BMAL
promoter-driven luciferase reporter in U2OS cells, we were able
to capture real time bioluminescent oscillations of synchronized
cells.9 A single end point assay of BMAL-luciferase suppression
by REV-ERBα agonists (including compound 1) has been
described in the literature.10 We confirmed that comparable
data (a statistically significant 15% suppression) can be
generated with compound 1 through our in-house U2OS
reporter assay. Further, we showed that the natural ligand for
REV-ERBα (heme) produced a >50% suppression in the same
assay (data not shown). A representative data set from the assay
for amine 4 is shown in Figure 4. Additional data from the assay
are also shown in Table 1 and Supporting Information Figure 2.
As can be seen, the increase in potency for recruitment of the
NCOR peptide promoted by compound 4 correlates with the
To assess the cellular efficacy of REV-ERBα activation and
selectivity over a LXRα driven pathway, amines 4, 10, 16, and
23 were profiled for their ability to inhibit LPS induction of IL-
6 production and to up-regulate expression of ABCA1 in
human THP-1 cells. All compounds demonstrated a significant
reduction of IL-6 secretion following treatment with 4, 10, 16,
and 23 at 1 μM with no measurable effect on ABCA1 levels
(Figure 6). Additionally, none of the compounds showed any
toxicity in measurement of ATP levels in THP-1 cells (see
Supporting Information Figure 3). These data indicated that
each of these tertiary amines was able to potently activate REV-
ERBα in cells, that suitable selectivity over LXRα had been
achieved, and that no general toxicity was skewing the results.
The iv and oral DMPK profiles of compounds 3, 4, 10, 16,
and 23 were evaluated in a 1 mg/kg cassette dose experiment in
C57Bl/6 mice. Four of the compounds demonstrated
essentially identical profiles with short half-lives, high clearance,
and low oral bioavailability. Compound 4 (GSK2945) was
differentiated from the group with a longer half-life of 2.0 h and
an oral bioavailability of 23%, despite the higher cLogP of the
compound (Table 2 and Supporting Information Figure 4).
The profile of amine 4 is suitable to achieve plasma
concentrations around the IC50 of the compound with oral
doses around 20−30 mg/kg, a dosing regimen that is
compatible with long-term in vivo studies. The remaining
four compounds (3, 10, 16, 23) would be suitable for acute
time-of-day dosing by injection where short exposure of the
compound at meaningful levels is desired. The specific
advantage of 10, 16, and 23 over the previously reported
tertiary amine 3 is the selectivity profile observed for REV-
ERBα over LXRα.
Figure 4. Oscillation of BMAL-Luc gene over time in synchronized
U2OS cells with and without treatment with compound 4.
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EXPERIMENTAL SECTION
■
General. All chemical reagents were purchased and used as
received. 1H NMR spectra were recorded on a Varian Unity 300,
Varian Unity Plus-400, Bruker Avance 400, or a Bruker Avance 500.
̈
̈
Chemical shifts are expressed in parts per million (ppm, δ units).
Coupling constants are in units of hertz (Hz). Splitting patterns
describe apparent multiplicities and are designated as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), or brs (broad).
The LCMS system used to determine purity was a UPLC analysis
conducted on a Waters Acquity system with a BEH C18, 2 mm × 50
mm, 1.7 μm column at 40 °C, 95% H2O, 5% MeCN to 99% MeCN in
1.1 min, holding at 100% MeCN for 40 s. Water contained 0.2% v/v
formic acid, and MeCN contained 0.15% v/v formic acid. The flow
rate was 1 mL/min with 5 μL of solution injected. Mass spectra were
recorded utilizing electrospray ionization (ESI) or atmospheric
pressure chemical ionization (APCI) switching between positive and
negative modes with DAD scanning from 210 to 350 nm. All
compounds tested were of ≥95% purity.
Normal phase chromatography was accomplished using either Isco
or Biotage equipment using prepacked silica gel columns.
Reverse phase HPLC was accomplished using Agilent 110 series
preparative HPLC systems using a C18 Phenomenex Luna, 75 mm ×
30 mm, 5 μm column using the gradient described. The flow rate was
70 mL/min, and the product was collected based on UV detection at
220 or 254 nm.
General Procedures for Reductive Amination. General
Method A (Used for Compounds 4, 10, 16, and 22−24).
Aldehyde (1.0 equiv) was added to a solution of amine (1.0 equiv) in
DCE (0.3 M) at room temperature. After the mixture was stirred for 5
min, acetic acid (1.0 equiv) and sodium triacetoxyborohydride (1.4
equiv) were added at room temperature. After the mixture was stirred
for 16 h, the second aldehyde (1.0 equiv) was added followed by an
additional portion of sodium triacetoxyborohydride (1.4 equiv). After
being stirred for 24 h at room temperature, the mixture was quenched
with saturated aqueous NaHCO3 solution. The organic phase was
dried over sodium sulfate, and the solvent removed under reduced
pressure.
Figure 6. Effects of compounds 4, 10, 16, and 23 on cellular makers of
(a) REV-ERBα and (b) LXRα activity. THP-1 cells were stimulated
with LPS, 10 μg/mL, in the presence of DMSO or compounds 4, 10,
16, 23 (1 μM), in triplicate. After 6 h the cells were lysed and the RNA
was analyzed using RT-qPCR. All compounds repressed IL-6 without
affecting ABCA1 transcript expression (∗, p < 0.05; ∗∗, p < 0.01; one-
way ANOVA with post hoc Dunnett’s).
General Method B (Used for Compounds 5−9, 11−15, and
25−26). MP-sodium cyanoborohydride (2.0 equiv) was weighed into
a 5 mL microwave reaction vessel. Amine (1.0 equiv) and aldehyde
(1.1 equiv) were dissolved in a mixture of CH2Cl2, EtOH, and AcOH
(2.5:2.5:1, 0.05 M) and then added to the reaction vessel. The mixture
was heated to 120 °C for 10 min using microwave irradiation. The
crude reaction was filtered, concentrated to an oil, dissolved in DMSO,
and purified by reverse phase preparative HPLC (20−100% MeCN in
water with 0.5% TFA) to give an oil. The oil was dissolved in CH2Cl2
and treated with 300 mg of MP-carbonate resin and filtered. The
filtrate was treated with 2 mL of 1 N HCl in MeOH and concentrated
to give the title compound.
General Method C (Used for Compounds 17−21). Aldehyde
(0.3 mmol) was weighed into a 2 mL microwave vial. In a 20 mL
scintillation vial, amine (0.3 mmol) was weighed, and to this 2 mL of a
3:3:1 CH2Cl2/EtOH/AcOH solution was added. The resulting
solution was added to the mixture along with polymer supported
sodium cyanoborohydride, 2 mM/g (4.77 mmol). The mixture was
heated to 120 °C in the microwave for 10 min. The mixture was
CONCLUSION
■
Herein we report on the discovery of REV-ERBα agonists from
the amine 1 series that demonstrated >1000-fold selectivity
over LXRα and have improved DMPK properties. In addition,
we have identified PGC1-β as a novel cofactor for REV-ERBα
and have noted that reported ligands 1−3 recruit different
cofactor peptides to REV-ERBα and may induce distinct
pharmacology as a result. Tertiary amine 4 is a potent agonist
with a profile suitable for chronic in vivo dosing via both oral
and iv routes. Other compounds (10, 16, and 23) are identified
with a shorter half-life in rodents that could be used for acute
time-of-day dosing studies. These compounds represent
valuable additions to the pharmacological toolbox to investigate
the biology of REV-ERBα without the complication of activity
on LXRα.
Table 2. Pharmacokinetic Parameters
dose route (1 mg/kg)
iv
PK parameter
3
4
10
16
23
CL (L h−1 kg−1
)
2.7
0.4
2.8
2.8
2.6
Vss (L/kg)
1.6
0.3
1.5
1.8
1.4
terminal t1/2 (h)
AUClast (h·ng/mL)
Tmax (h)
0.55
365
0.50
7.41
7.93
2.2
1.65
2,538
2.0
0.74
354
0.25
8.98
8.39
2.4
1.27
361
0.25
9.14
10.2
3.5
0.74
375
0.25
9.94
9.39
3.0
oral
Cmax (ng/mL)
AUClast (h·ng/mL)
F (%)
180
594
23.4
E
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filtered through a disposable filter funnel to remove the sodium
cyanoborohydride and evaporated overnight under reduced pressure.
The resulting solid was dissolved in DMSO and purified by reverse
phase preparative HPLC (20−100% MeCN in water with 0.5% TFA).
N-(4-Chloro-2-methylbenzyl)-N-(4-chlorobenzyl)-1-(5- nitro-
thiophen-2-yl)methanamine (4). By use of general method A, the
title compound was synthesized using 4-chloro-2-methylbenzaldehyde,
5-nitrothiophene-2-carbaldehyde, and 4-chlorobenzylamine. The res-
idue was purified over silica gel (10−30% EtOAc in petroleum ether)
to give the title compound (0.42 g, 1.0 mmol, 50% yield) as a pale
The residue was purified over silica gel (10−30% EtOAc in petroleum
ether) and ISOLUTE SCX-2 SPE column (NH3 solution, 2 N in
MeOH) to give the title compound (0.29 g, 0.8 mmol, 40%) as a pale
1
yellow oil. H NMR (400 MHz, CDCl3) δ 8.61−8.55 (m, 1H), 8.52
(dd, J = 4.8, 1.7 Hz, 1H), 7.82−7.69 (m, 2H), 7.40−7.24 (m, 3H),
7.08−6.98 (m, 2H), 6.87−6.84 (m, 1H), 3.74 (brs, 2H), 3.64 (brs,
2H), 3.60 (brs, 2H). 13C NMR (DEPTQ-135) (101 MHz, CDCl3) δ
162.5 (d, J = 246.4 Hz), 153.4, 151.0, 150.2, 149.3, 136.4, 133.6, 133.5
(d, J = 3.3 Hz), 130.3 (d, J = 7.9 Hz), 128.8, 124.5, 123.7, 115.6 (d, J =
21.7 Hz), 57.5, 55.4, 52.9. 19F NMR (377 MHz, CDCl3) δ −114.6 to
−114.7 (m, 1F); 19F{H} NMR (377 MHz, CDCl3) δ −114.6 (s, 1F).
LCMS: tR = 1.95 min, 100%. MS m/z = 358.1 (M + H)+. HRMS calcd
for C18H17O2N3FS (M + H)+ requires m/z 358.1020, found 358.1019.
N-(2-Methylbenzyl)-1-(5-nitrothiophen-2-yl)-N-(pyridin-3-
ylmethyl)methanamine Hydrochloride Salt (11). By use of
general method B, the title compound was afforded as a white solid
using 2-methylbenzaldehyde and amine 28 (31 mg, 66 μmol, 28%). 1H
NMR (400 MHz, MeOD) δ 8.62−8.60 (m, 1H), 8.58−8.56 (m, 1H),
8.35 (d, J = 8.2 Hz, 1H), 7.83−7.79 (m, 2H), 7.34−7.32 (m, 1H),
7.11−7.07 (m, 3H), 7.02 (d, J = 4.1 Hz, 1H), 3.92 (brs, 2H), 3.85 (brs,
2H), 3.73 (brs, 2H), 2.30 (s, 3H). LCMS: tR = 0.79 min, 100%. MS m/
z = 354 (M + H)+. HRMS calcd for C19H20N3O2S (M + H)+ requires
354.1276, found 354.1278.
1
yellow oil. H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 4.2 Hz, 1H),
7.37−7.29 (m, 5H), 7.18−7.11 (m, 2H), 6.86−6.81 (m, 1H), 3.70
(brs, 2H), 3.59 (brs, 2H), 3.55 (d, J = 0.8 Hz, 2H), 2.26 (s, 3H). 13C
NMR (j-mod) (101 MHz, CDCl3) δ 153.4, 151.0, 139.2, 136.4, 134.5,
133.5, 133.1, 130.8, 130.5, 130.3, 128.8, 128.7, 126.2, 124.6, 58.1, 55.7,
53.0, 19.4. LCMS: tR = 1.60 min, 98%. MS m/z = 421.1 (M + H)+.
HRMS calcd for C20H19N2O2Cl2S (M + H)+ requires m/z 421.0539,
found 421.0536.
N-Benzyl-N-(4-chlorobenzyl)-1-(5-nitrothiophen-2-yl)-
methanamine (5). By use of general method B, the title compound
was afforded as a white solid using amine 27 and benzaldehyde (27
1
mg, 59 μmol, 28%). H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 4.1
Hz, 1H), 7.44−7.28 (m, 9H), 6.94−6.86 (m, 1H), 3.78 (brs, 2H),
3.73−3.62 (m, 4H). LCMS: tR = 1.21 min, 100%. MS m/z = 373 (M +
H)+. HRMS calcd for C19H18ClN2O2S (M + H)+ requires 373.0778,
found 373.0780.
N-(3-Methylbenzyl)-1-(5-nitrothiophen-2-yl)-N-(pyridin-3-
ylmethyl)methanamine Hydrochloride Salt (12). By use of
general method B, the title compound was afforded as a white solid
using 3-methylbenzaldehyde and amine 28 (34 mg, 73 μmol, 31%). 1H
NMR (400 MHz, MeOD) δ 8.72−8.70 (m, 1H), 8.62−8.61 (m, 1H),
8.44 (d, J = 8.2 Hz, 1H), 7.86−7.83 (m, 2H), 7.40−7.30 (m, 1H),
7.19−7.17 (m, 2H), 7.06−7.04 (m, 1H), 7.02−7.01 (m, 1H), 3.89
(brs, 2H), 3.85 (brs, 2H), 3.67 (brs, 2H), 2.30 (s, 3H). LCMS: tR =
0.81 min, 100%. MS m/z = 354 (M + H)+. HRMS calcd for
C19H20N3O2S (M + H)+ requires 354.1271, found 354.1271.
N-(4-Methoxybenzyl)-1-(5-nitrothiophen-2-yl)-N-(pyridin-3-
ylmethyl)methanamine Hydrochloride Salt (13). By use of
general method B, the title compound was afforded as a white solid
using 4-methoxybenzaldehyde and amine 28 (24 mg, 49 μmol, 21%).
1H NMR (400 MHz, MeOD) δ 8.71−8.70 (m, 1H), 8.75−8.64 (m,
1H), 8.62−8.55 (m, 1H), 8.45−8.40 (m, 1H), 7.40−7.25 (m, 2H),
7.30−7.21 (m, 2H), 7.01 (d, J = 4.3 Hz, 1H), 6.40−6.36 (m, 1H), 3.88
(brs, 2H) 3.83 (brs, 2H), 3.74 (s, 3H), 3.65 (brs, 2H). LCMS: tR =
0.75 min, 100%. MS m/z = 370 (M + H)+. HRMS calcd for
C19H20N3O3S (M + H)+ requires 370.1225, found 370.1229.
N-(3-Methoxybenzyl)-1-(5-nitrothiophen-2-yl)-N-(pyridin-3-
ylmethyl)methanamine Hydrochloride Salt (14). By use of
general method B, the title compound was afforded as a white solid
using 3-methoxybenzaldehyde and amine 28 (36 mg, 75 μmol, 32%).
1H NMR (400 MHz, MeOD) δ 8.73−8.71 (m, 1H), 8.62−8.61 (m,
1H), 8.45−8.43 (m, 1H), 7.86−7.83 (m, 2H), 7.25−7.16 (m, 1H),
7.02 (d, J = 4.1 Hz, 1H), 6.98−6.92 (m, 2H), 6.81−6.77 (m, 1H), 3.90
(brs, 2H), 3.86 (brs, 2H), 3.77 (s, 3H), 3.68 (brs, 2H). LCMS: tR =
0.75 min, 100%. MS m/z = 370 (M + H)+. HRMS calcd for
C19H20N3O3S (M + H)+ requires 370.1225, found 370.1220.
N-(3-Trifluromethylbenzyl)-1-(5-nitrothiophen-2-yl)-N-(pyri-
din-3-ylmethyl)methanamine Hydrochloride Salt (15). By use of
general method B, the title compound was afforded as a white solid
using 3-trifluoromethylbenzaldehyde and amine 28 (21 mg, 40 μmol,
17%). 1H NMR (400 MHz, MeOD) δ 8.71−8.68 (m, 1H), 8.62−8.58
(m, 1H), 8.35−8.32 (m, 1H), 8.23 (brs, 1H), 7.91−7.74 (m, 2H),
7.69−7.64 (m, 2H), 7.57−7.48 (m, 2H), 7.06 (d, J = 4.1 Hz, 1H), 3.90
(brs, 2H), 3.86 (brs, 2H), 3.80 (brs, 2H). LCMS: tR = 0.84 min, 100%.
MS m/z = 408 (M + H)+. HRMS calcd for C19H17F3N3O2S (M + H)+
requires 408.0994, found 408.0997.
5-(((4-Chlorobenzyl)(pyridin-3-ylmethyl)amino)methyl)-
thiophene-2-carbonitrile (16). By use of general method A, the title
compound was synthesized using 3-pyridinecarboxaldehyde, 5-
formylthiophene-2-carbonitrile, and 4-chlorobenzylamine. The residue
was purified over silica gel (30−70% EtOAc in petroleum ether) and
ISOLUTE SCX-2 SPE column (NH3 solution, 2 N in MeOH) to give
the title compound (0.43 mg, 1.2 mmol, 61% yield) as a pale yellow
N-(4-Chlorobenzyl)-N-(2-fluorobenzyl)-1-(5-nitrothiophen-
2-yl)methanamine Hydrochloride Salt (6). By use of general
method B, the title compound was afforded as a white solid using
1
amine 27 and 2-fluorobenzaldehyde (20 mg, 47.0 μmol, 22%). H
NMR (400 MHz, MeOD) δ 7.82 (d, J = 4.3 Hz, 1H), 7.52 (dd, J = 7.5,
1.2 Hz, 1H), 7.49−7.36 (m, 2H), 7.34−7.27 (m, 3H), 7.20−7.16 (m,
1H), 7.09−7.04 (m, 1H), 6.99 (d, J = 4.3 Hz, 1H), 3.79 (s, 2H), 3.72
(s, 2H), 3.64 (s, 2H). LCMS: tR = 1.25 min, 100%. MS m/z = 391 (M
+ H)+. HRMS calcd for C19H17ClFN2O2S (M + H)+ requires
391.0683, found 391.0684.
N-(4-Chlorobenzyl)-3-methyl-N-((5-nitrothiophen-2-yl)-
methyl)butan-1-amine Hydrochloride Salt (7). By use of general
method B, the title compound was afforded as a white solid using
1
amine 27 and 3-methylbutanal (13 mg, 36.0 μmol, 18%). H NMR
(400 MHz, DMSO-d6) δ 11.7 (brs, 1H), 8.12−8.10 (m, 1H), 7.79−
7.36 (m, 5H), 4.71−4.51 (m, 1H), 4.48−4.25 (m, 2H), 3.12−3.02 (m,
1H), 2.80−2.30 (m, 2H), 1.77−1.38 (m, 3H), 0.80 (d, J = 6.5 Hz,
6H). LCMS: tR = 1.13 min, 100%. MS m/z = 353 (M + H)+. HRMS
calcd for C17H22ClN2O2S (M + H)+ requires 353.1091, found
353.1091.
N-(4-Chlorobenzyl)-1-(5-nitrothiophen-2-yl)-N-(pyridin-3-
ylmethyl)methanamine Hydrochloride Salt (8). By use of general
method B, the title compound was afforded as a white solid using
amine 27 and 3-pyridylcarbaldehyde (27 mg, 59 μmol, 28%). 1H NMR
(400 MHz, CDCl3) δ 12.15 (brs, 1H), 8.71−8.70 (m, 1H), 8.62−8.60
(m, 1H), 8.19−8.16 (m, 1H), 7.73−7.63 (m, 2H), 7.28−7.19 (m, 4H),
6.84 (d, J = 4.3 Hz, 1H), 3.73 (s, 2H), 3.70 (s, 2H), 3.57 (s, 2H).
LCMS: tR = 0.81 min, 95%. MS m/z = 374 (M + H)+. HRMS calcd for
C18H17ClN3O2S (M + H)+ requires 374.0730, found 374.0731.
N-(4-Methylbenzyl)-1-(5-nitrothiophen-2-yl)-N-(pyridin-3-
ylmethyl)methanamine Hydrochloride Salt (9). By use of general
method B, the title compound was afforded as a white solid using
amine 28 and 4-methylbenzaldehyde (35 mg, 75 μmol, 32%). 1H
NMR (400 MHz, MeOD) δ 8.72−8.70 (m, 1H), 8.62 (d, J = 6.1 Hz,
1H), 8.44−8.42 (m, 1H), 7.85−7.83 (m, 2H), 7.25−7.24 (m, 2H),
7.13−7.11 (m, 2H), 7.02−7.01 (m, 1H), 3.88 (brs, 2H), 3.83 (brs,
2H), 3.66 (brs, 2H), 2.27 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
154.7, 151.8, 149.8, 146.6, 145.6, 136.6, 134.7, 130.3, 130.0, 129.3,
129.1, 128.7, 125.8, 57.0, 54.1, 52.1, 20.7. LCMS: tR = 0.82 min, 100%.
MS m/z = 354 (M + H)+. HRMS calcd for C19H20N3O2S (M + H)+
requires 354.1276, found 354.1278.
N-(4-Fluorobenzyl)-1-(5-nitrothiophen-2-yl)-N-(pyridin-3-
ylmethyl)methanamine (10). By use of general method A, the title
compound was synthesized using 4-fluorobenzaldehyde and amine 28.
F
dx.doi.org/10.1021/jm400458q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
oil. 1H NMR (400 MHz, CDCl3) δ 8.61−8.55 (m, 1H), 8.53 (dd, J =
4.8, 1.5 Hz, 1H), 7.75−7.69 (m, 1H), 7.46 (d, J = 3.8 Hz, 1H), 7.36−
7.27 (m, 5H), 6.92−6.89 (m, 1H), 3.77 (brs, 2H), 3.62 (brs, 2H), 3.59
(brs, 2H). 13C NMR (DEPTQ-135) (101 MHz, CDCl3) δ 152.2,
150.2, 149.2, 137.6, 136.5, 136.3, 133.7, 133.5, 130.0, 128.9, 125.5,
123.7, 114.5, 109.0, 57.4, 55.3, 52.4. LCMS: tR = 1.00 min, 98%. MS
338.1 (M + H)+. HRMS C19H17N3FS (M + H)+ requires m/z
338.1122, found 338.1128.
5-(((3,4-Difluorobenzyl)(pyridin-3-ylmethyl)amino)methyl)-
thiophene-2-carbonitrile (23). By use of general method A, the title
compound was synthesized using 3,4-difluorobenzaldehyde, 5-
formylthiophene-2-carbonitrile, and 3-(aminomethyl)pyridine. The
residue was purified over silica gel (30−70% EtOAc in petroleum
ether) and ISOLUTE SCX-2 SPE column (NH3 solution, 2 N in
MeOH) to give the title compound (0.42 g, 1.2 mmol, 59% yield) as a
+
m/z = 354.1 (M + H)+. HRMS calcd for C19H17N3ClS (M + H)
requires m/z 354.0826, found 354.0824.
1-(4-Bromo-5-methylthiophen-2-yl)-N-(4-chlorobenzyl)-N-
(pyridin-3-ylmethyl)methanamine (17). By use of general method
C, the title compound was afforded using 4-bromo-5-methylthio-
phene-2-carbaldehyde and [(4-chlorophenyl)methyl] (3-
1
pale yellow oil. H NMR (400 MHz, CDCl3) δ 8.59−8.53 (m, 1H),
8.51 (dd, J = 4.8, 1.7 Hz, 1H), 7.74−7.68 (m, 1H), 7.45 (d, J = 3.8 Hz,
1H), 7.28 (ddd, J = 7.9, 4.8, 40.5 Hz, 1H), 7.24−7.15 (m, 1H), 7.15−
7.04 (m, 2H), 6.93−6.88 (m, 1H), 3.77 (brs, 2H), 3.61 (brs, 2H), 3.56
(brs, 2H). 13C NMR (DEPTQ-135) (101 MHz, CDCl3) δ 151.8,
150.5 (dd, J = 248.9, 12.9 Hz), 150.1, 149.8 (dd, J = 248.0, 12.8 Hz),
149.2, 137.6, 136.3, 135.2 (dd, J = 5.2, 3.8 Hz), 133.4, 125.6, 124.5
(dd, J = 6.4, 3.6 Hz), 123.7, 117.3 (dd, J = 17.1, 7.5 Hz), 114.3, 109.0,
57.0, 55.3, 52.4, one carbon missing. 19F NMR (377 MHz, CDCl3) δ
−139.3 to −139.1 (m, 1F), −137.2 (ddd, J = 21.8, 11.0, 6.8 Hz, 1F).
19F{H} NMR (377 MHz, CDCl3) δ −139.2 (d, J = 20.3 Hz, 1F),
1
pyridinylmethyl)amine as a white solid (72 mg, 170 μmol, 54%). H
NMR (400 MHz, DMSO-d6) δ 8.68−8.64 (m, 1H), 8.61−8.57 (m,
1H), 8.16−8.11 (m, 1H), 7.68−7.62 (m, 1H), 7.40−7.34 (m, 4H),
6.92−6.89 (m, 1H), 3.71−3.63 (m, 4H), 3.57 (brs, 2H), 2.29 (s, 3H).
LCMS: tR = 0.98 min, 100%. MS m/z = 421 (M + H)+. HRMS calcd
for C19H19BrClN2S (M + H)+ requires 421.0141, found 421.0139.
1-(Benzo[b]thiophen-2-yl)-N-(4-chlorobenzyl)-N-(pyridin-3-
ylmethyl)methanamine (18). By use of general method C, the title
compound was afforded using benzo[b]thiophene-2-carbaldehyde and
[(4-chlorophenyl)methyl](3-pyridinylmethyl)amine as a white solid
(24 mg, 62 μmol, 12%). 1H NMR (400 MHz, DMSO-d6) δ 8.67−8.62
(m, 1H), 8.55−8.51 (m, 1H), 8.00−7.91 (m, 2H), 7.79−7.73 (m, 1H),
7.54−7.38 (m, 5H), 7.36−7.28 (m, 3H), 3.85 (brs, 2H), 3.68 (brs,
2H), 3.63 (brs, 2H). LCMS: tR = 0.93 min, 100%. MS m/z = 379 (M +
H)+. HRMS calcd for C22H20ClN2S (M + H)+ requires 379.1036,
found 379.1038.
−137.2 (d, J = 20.3 Hz, 1F). LCMS: tR = 0.92 min, 100%. MS m/z =
356.1 (M + H)+. HRMS calcd for C19H16N3F2S requires m/z
356.1028, found 356.1032.
5-(((4-Methoxybenzyl)(pyridin-3-ylmethyl)amino)methyl)-
thiophene-2-carbonitrile (24). By use of general method A, the title
compound was synthesized using 3-pyridinecarboxaldehyde, 5-
formylthiophene-2-carbonitrile, and 4-methoxybenzylamine. The
residue was purified over silica gel (20−70% EtOAc in petroleum
ether) and ISOLUTE SCX-2 SPE column (NH3 solution, 2 N in
MeOH) to give the title compound (0.17 g, 0.5 mmol, 50% yield) as a
1-(3-(1H-Pyrrol-1-yl)thiophen-2-yl)-N-(4-chlorobenzyl)-N-
(pyridin-3-ylmethyl)methanamine (19). By use of general method
C, the title compound was afforded using 3-(1H-pyrrol-1-yl)-2-
thiophenecarbaldehyde and [(4-chlorophenyl)methyl](3-
1
pale yellow oil. H NMR (400 MHz, CDCl3) δ 8.62−8.56 (m, 1H),
8.52 (dd, J = 4.8, 1.5 Hz, 1H), 7.78−7.72 (m, 1H), 7.46 (d, J = 3.8 Hz,
1H), 7.32−7.25 (m, 3H), 6.92−6.86 (m, 3H), 3.80 (s, 3H), 3.76 (brs,
2H), 3.62 (brs, 2H), 3.57 (brs, 2H). LCMS: tR = 0.89 min, 100%. MS
m/z = 350.1 (M + H)+. HRMS calcd for C20H20ONS requires m/z
350.1322, found 350.1327.
1
pyridinylmethyl)amine as a white solid (44 mg, 110 μmol, 35%). H
NMR (400 MHz, DMSO-d6) δ 8.61−8.60 (m, 2H), 8.06−8.03 (m,
1H), 7.66−7.63 (m, 1H), 7.54 (d, J = 5.5 Hz, 1H), 7.36−7.31 (m,
4H), 7.06 (d, J = 5.5 Hz, 1H), 6.94−6.93 (m, 2H), 6.17 (t, J = 2.1 Hz,
2H), 3.71 (brs, 2H), 3.60 (brs, 2H), 3.54 (brs, 2H). LCMS: tR = 0.90
min, 100%. MS m/z = 394 (M + H)+. HRMS calcd for C22H21ClN3S
(M + H)+ requires 394.1147, found 394.1145.
5-(((2,5-Difluorobenzyl)(pyridin-3-ylmethyl)amino)methyl)-
thiophene-2-carbonitrile (25). By use of general method A, the title
compound was synthesized using 2,5-difluorobenzaldehyde, 5-
formylthiophene-2-carbonitrile, and N-methyl-1-(pyridin-3-yl)-
methanamine as a white solid (23 mg, 60 μmol, 31%). 1H NMR
(400 MHz, MeOD) δ 8.92−8.91 (m, 1H), 8.79−8.77 (m, 1H), 8.73−
8.71 (m, 1H), 8.08−8.04 (m, 1H), 7.61−7.59 (m, 1H), 7.31−7.30 (m,
1H), 7.29−7.27 (m, 1H), 7.25−7.00 (m, 2H), 4.17 (brs, 2H), 4.11
(brs 2H), 3.93 (brs, 2H). LCMS: tR = 0.77 min, 100%. MS m/z = 356
(M + H)+. HRMS calcd for C19H16F2N3S (M + H)+ requires
356.1033, found 356.1037.
N-(4-Chlorobenzyl)-1-(pyridin-3-yl)-N-(thiazol-2-ylmethyl)-
methanamine (20). By use of general method C, the title compound
was afforded using 1,3-thiazole-2-carbaldehyde and [(4-chlorophenyl)-
methyl](3-pyridinylmethyl)amine as a white solid (53 mg, 160 μmol,
1
51%). H NMR (400 MHz, DMSO-d6) δ 8.72−8.66 (m, 1H), 8.64−
8.59 (m, 1H), 8.19−8.12 (m, 1H), 7.76−7.63 (m, 3H), 7.44−7.34 (m,
4H), 3.90 (brs, 2H), 3.75 (brs, 2H), 3.66 (brs, 2H). LCMS: tR = 0.67
min, 95%. MS m/z = 330 (M + H)+. HRMS calcd for C17H17ClN3S
(M + H)+ requires 330.0832, found 330.0836.
5-(((2-Fluorobenzyl)(pyridin-3-ylmethyl)amino)methyl)-
thiophene-2-carbonitrile (26). By use of general method A, the title
compound was synthesized using 2-fluorobenzaldehyde, 5-formylth-
iophene-2-carbonitrile, and 3-aminomethylpyridine as a white solid
N-((1H-Imidazol-2-yl)methyl)-N-(4-chlorobenzyl)-1-(pyridin-
3-yl)methanamine (21). By use of general method C, the title
compound was afforded using 1H-imidazole-2-carbaldehyde and [(4-
chlorophenyl)methyl](3-pyridinylmethyl)amine as a white solid (47
1
(35 mg, 94 μmol, 50%). H NMR (400 MHz, MeOD) δ 9.00−8.99
(m, 1H), 8.85−8.76 (m, 2H), 8.08−8.05 (m, 1H), 7.67−7.61 (m, 1H),
7.58−7.50 (m, 1H), 7.40−7.30 (m, 2H), 7.21−7.16 (m, 1H), 7.14−
7.03 (m, 1H), 4.38 (brs, 2H), 4.31 (brs, 2H), 4.12 (brs, 2H). LCMS:
tR = 0.53 min, 100%. MS m/z = 338 (M + H)+. HRMS calcd for
C19H17FN3S (M + H)+ requires 338.1129, found 338.1127.
1
mg, 150 μmol, 47%). H NMR (400 MHz, MeOD) δ 8.77−8.76 (m,
1H), 8.64−8.62 (m, 1H), 8.41−8.38 (m, 1H), 7.82−7.79 (m, 1H),
7.37 (s, 2H), 7.31−7.25 (m, 4H), 4.01 (brs, 2H), 3.89 (brs, 2H), 3.70
(brs, 2H). LCMS: tR = 0.43 min, 100%. MS m/z = 313 (M + H)+.
HRMS calcd for C17H18ClN4 (M + H)+ requires 313.1220, found
313.1220.
N-(4-Chlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine
(27). 5-Nitrothiophene-2-carboxaldehyde (0.57 g, 3.6 mmol) was
added to a solution of 4-chlorobenzylamine (0.44 mL, 3.6 mmol) in
DCE (12.0 mL) at room temperature. After the mixture was stirred for
5 min, acetic acid (0.22 mL, 3.6 mmol) and sodium triacetoxybor-
ohydride (1.10 g, 5.0 mmol) were added at room temperature. After
being stirred for 16 h at room temperature, the mixture was quenched
with saturated aqueous NaHCO3 solution. The organic phase was
dried over sodium sulfate, and the solvent was evaporated in vacuo.
The residue was purified over silica gel (20−60% EtOAc in petroleum
ether) to give the title compound as a pale yellow oil (0.55 g, 1.9
mmol, 54%). 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 4.2 Hz, 1H),
7.32−7.26 (m, 4H), 6.86 (dt, J = 4.2, 1.0 Hz, 1H), 3.98 (d, J = 1.0 Hz,
5-(((4-Fluorobenzyl)(pyridin-3-ylmethyl)amino)methyl)-
thiophene-2-carbonitrile (22). By use of general method A, the title
compound was synthesized using 3-pyridinecarboxaldehyde, 5-
formylthiophene-2-carbonitrile, and 4-fluorobenzylamine. The residue
was purified over silica gel (20−80% EtOAc in petroleum ether) and
ISOLUTE SCX-2 SPE column (NH3 solution, 2 N in MeOH) to give
the title compound (0.17 g, 0.51 mmol, 51% yield) as a pale yellow oil.
1H NMR (500 MHz, CDCl3) δ 8.63−8.57 (m, 1H), 8.57−8.51 (m,
1H), 7.80−7.75 (m, 1H), 7.47 (d, J = 3.7 Hz, 1H), 7.37−7.30 (m,
3H), 7.07−7.00 (m, 2H), 6.92−6.89 (m, 1H), 3.78 (brs, 2H), 3.63
(brs, 2H), 3.59 (brs, 2H). LCMS: tR = 0.89 min, 100%. MS m/z =
G
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2H), 3.82 (brs, 2H). MS m/z = 283.0 (M + H)+. HRMS calcd for
C12H11O2N2ClS requires m/z 283.0303, found 283.0306.
assay plates containing 100 nL of test compound. The plate was
incubated for 1 h at room temperature and read on ViewLux in Lance
mode for EU/APC. Raw data were analyzed using ABASE (IBDS)
software. The data were normalized initially using the following
equation: normalization =100 × [(basal HTRF − value)/(basal HTRF
− minimal HTRF)]. The normalized data were then was fit to a four-
parameter logistic equation.
1-(5-Nitrothiophen-2-yl)-N-(pyridin-3-ylmethyl)-
methanamine (28). 5-Nitrothiophene-2-carboxaldehyde (2.28 g,
14.4 mmol) was added to a solution of 3-aminomethylpyridine
(1.46 mL, 14.4 mmol) in DCE (48.0 mL) at room temperature. After
the mixture was stirred for 5 min, acetic acid (0.86 mL, 14.4 mmol)
and sodium triacetoxyborohydride (4.40 g, 20.0 mmol) were added at
room temperature. After being stirred for 16 h at room temperature,
the mixture was quenched with saturated aqueous NaHCO3 solution.
The organic phase was dried over sodium sulfate, and the solvent was
evaporated in vacuo. The residue was purified over silica gel (20−70%
EtOAc in petroleum ether) to give the title compound as a brown gum
(2.26 g, 9.0 mmol, 63% yield). 1H NMR (400 MHz, CDCl3) δ 8.57−
8.51 (m, 1H), 8.50 (dd, J = 4.8, 1.8 Hz, 1H), 7.76 (d, J = 4.2 Hz, 1H),
7.70 (dt, J = 7.8, 1.8 Hz, 1H), 7.26 (ddd, J = 7.8, 4.8, 0.5 Hz, 1H), 6.86
(ddd, J = 4.2, 1.0 Hz, 1H), 3.99 (d, J = 1.0 Hz, 2H), 3.86 (brs, 2H).
MS m/z = 250.1 (M + H)+. HRMS calcd for C11H11O2N3S requires
m/z 250.0645, found 250.0641.
5-(((Pyridin-3-ylmethyl)amino)methyl)thiophene-2-carboni-
trile (29). 3-Aminomethylpyridine (1.51 g, 14.0 mmol) and 5-formyl-
2-thiophenecarbonitrile (1.60 g, 11.7 mmol) were dissolved in
anhydrous THF (40 mL) and acetic acid (10 mL) to give a clear
solution. The mixture was stirred at room temperature for 30 min.
Sodium triacetoxyborohydride (2.97 g, 14 mmol) was added, and the
mixture was stirred at room temperature for 1 h. The mixture was
concentrated to an oil which was purified over silica gel (2−10%
MeOH in CH2Cl2) to give the title compound as an oil (1.34 g, 0.6
mmol, 50% yield) . 1H NMR (400 MHz, DMSO-d6) δ 10.9 (brs, 1H),
9.17−9.11 (m, 1H), 8.98−8.85 (m, 1H), 8.80−8.72 (m, 1H), 8.11−
8.02 (m, 1H), 7.95 (d, J = 4.2 Hz, 1H), 7.60 (d, J = 4.2 Hz, 1H), 4.54
(brs, 2H), 4.41 (brs, 2H). LCMS: tR = 0.24 min, 100%. MS m/z = 230
(M + H)+.
REV-ERBα NCOR FRET Assay. REV-ERBα was made at GSK.
Proteins were chemically biotinylated using standard methods.
Biotinylated NCOR1 was purchased from CPC Scientific. Streptavi-
din-labeled APC (CR130-150) and Eu-W1024 labeled streptavidin
(AD0063) were purchased from Perkin-Elmer. Compounds were
diluted in 100% neat DMSO at 10 mM. The compounds were
dispensed into an intermediate plate (polypropylene Greiner PP V-
bottom, 781280) to make serial dilutions in 100% neat DMSO.
Approximately 100 nL of the serial dilution was added to the assay
plate. The stock buffer used was a 0.5 M solution of MOPS made by
adding 104 g of MOPS to 800 mL of H2O in a graduated cylinder,
using a calibrated pH meter, and adding increasing amounts of NaOH
to give a final pH of 7.5. This solution was filtered using a Costar 0.2
μm filtering apparatus. The assay buffer used was prepared by adding
100 mL of 10× MOPS stock solution into a graduated cylinder to 800
mL. NaF (2.09 g), CHAPS (0.03 g), and BSA (0.1 g) were added to
the flask. Water was added to give a final volume of 1 L. The assay
buffer was filtered with a Costar 0.2 μm filtering apparatus. DTT was
added to the assay buffer to a final concentration of 10 mM. To a
polypropylene Costar conical centrifuge tube were added assay buffer
and an appropriate amount of biotinylated-NCOR1 from the 100 μM
stock solution to give a final concentration of 20 nM. To the above
biotinylated NCOR1 solution, an appropriate amount of europium-
labeled streptavidin was added to give a final concentration of 10 nM.
The solution was incubated for 15 min at room temperature. In a
separate polypropylene tube was added an appropriate amount of
biotinylated REV-ERBα protein from the stock solution to give a final
concentration of 20 nM. To the biotinylated-REV-ERBα solution was
added an appropriate amount of APC-labeled streptavidin to give a
final concentration of 10 nM. The resulting solution was incubated for
15 min at room temperature. A 20-fold excess of biotin from the 10
mM stock solution was added, and the resulting solution was
incubated for 10 min at room temperature. The above solutions were
gently mixed together to give a final solution containing 20 nM REV-
ERBα 10 nM APC, and 20 nM NCOR1_10 nM SA_EU. The
resulting mixture was incubated for 5 min, and a Thermo Combi
Multidrop was used to add 10 μL of peptide/REV-ERBα solution to
Peptide Scan. By use of the same reagents as for the NCOR FRET
assay, various biotinylated peptides were added to the Greiner assay
plate to give a final concentration of 20 nM. An appropriate amount of
Eu-W1024 was added to the plate to give a final concentration of 20
nM. The plate was incubated for 20 min at room temperature. At the
same time, in a polypropylene tube was added an appropriate amount
of biotinylated-REV-ERBα protein from the stock solution to give a
final concentration of 20 nM. To the biotinylated-REV-ERBα solution,
an appropriate amount of APC-labeled streptavidin was added to give
a final concentration of 10 nM. The tube was inverted gently to mix
and was incubated for 20 min at room temperature. Following the 20
min incubations, 20-fold excess biotin from the 10 mM stock solution
was added, and the tube was inverted gently to mix. After a 15 min
incubation at room temperature, a Thermo Combi Multidrop was
used to add 5 μL of the REV-ERBα solution to the plates containing
100 nL of the various peptides. The plates were read on a ViewLux in
Lance mode for EU/APC. The raw data were analyzed using an Excel
template that averaged the four wells of each peptide and generated a
standard deviation.
BMAL Reporter Gene Assay. Stable U2OS cell line expressing
mouse BMAL-promoter luciferase was generated using a published
procedure.9 Freshly passaged cells were seeded at confluence in 384-
well format. Robust circadian rhythms were induced by change of
medium, and synchronization was evaluated by measuring the clock-
driven luciferase reporter activity over 48 h. Synchronized cells were
treated with compound, and real time bioluminescent oscillations were
recorded for 72 h with measurements every 20 min using the
temperature controlled luminometer Orion II. After 72 h of treatment,
cells were washed and allowed to recover in full growth medium. The
recovery oscillations are monitored and used as an internal toxicity
counterscreen for off-target effects on cell transcription.
Cell Assays. THP-1 cells were obtained from ECACC. THP-1 cells
were subcultured between 3 × 105 and 8 × 105 cells/mL in RPMI1640
(PAA) supplemented with 10% FBS (Invitrogen U.K.). Total RNA
was prepared using the RNAeasy kit (Qiagen, U.K.) and treated with
DNASe (Qiagen, U.K.). Reverse transcription was performed using a
high capacity RNA to cDNA kit (Applied Biosystems, U.S.). The
cDNA was analyzed by qPCR using Power Sybr Green master mix
(Applied Biosystems, U.S.) and prevalidated Quantitect primers
(Qiagen, U.K.). TATA box binding protein was the housekeeping
gene. The cell viability assay was performed using the CellTiter-Glo kit
(Promega, U.S.). The kit was used according to the manufacturer’s
instructions. Briefly, cells were incubated for 6 h with the chemical
compounds. CellTiter-Glo was then added for 15 min before the
luminescence was recorded.
DMPK Experiment. Plasma levels of a cassette of the five test
compounds were evaluated in C57BL6 mice purchased from SLAC
Laboratory Animal Co. (male mice, 22−25 g, n = 3 per time point,
total of 9 animals per dosing regimen) administered by either iv tail
vein injection (1 mg/kg) or po oral gavage administration (1 mg/kg).
Compounds were dissolved in a 10% DMSO, 10% Solutol-HS-15, and
80% water solution. At 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h after
dosing, blood was taken. Plasma was generated using standard
centrifugation techniques and was frozen at −78 °C. Plasma was mixed
with acetonitrile (1:15 (v/v)) and vortexed for 2 min and centrifuged
at 14 000 rpm for 5 min. A 2 mL aliquot of the supernatant was
analyzed for drug levels by liquid chromatography/tandem mass
spectrometry.
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ASSOCIATED CONTENT
* Supporting Information
Time course data, peptide recruitment scans, toxicity data,
pharmacokinetics, and NMR spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: 919-483-9539. E-mail: ryan.p.trump@gsk.com.
■
Present Address
#M.B.: King Abdullah International Medical Research Center,
King Saud Bin Abdulaziz University for Health Sciences,
National Guard Health Affairs, P.O. Box 22490, Riyadh 11426,
Saudi Arabia.
Author Contributions
The manuscript was written with contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge G. Bruce Wisely and Tom G. Consler for
production of the REV-ERBα protein and Liz Clarke, William J.
Cairns, Emma Watts, and Danuta E. Mossakowa for aid in the
development of the U2OS BMAL reporter cell line. The
EPSRC is thanked for financial support, and the Mass
Spectrometry Service Centre, Swansea, U.K., is thanked for
high-resolution spectra.
ABBREVIATIONS USED
■
REV-ERBα, NR1D1; LXRα, liver X receptor α (NR1H3);
NCOR, nuclear receptor co-repressor 1; SMRT, nuclear
receptor co-repressor 2; PGC1-β, peroxisome proliferator-
activated receptor γ coactivator 1-β; IL-6, interleukin 6; THP-1,
human acute monocytic leukemia cell line; LPS, lipopolysac-
charide; ABCA1, ATP-binding cassette, subfamily A, member
1; GAL4, regulatory protein; BMAL, brain and muscle aryl
hydrocarbon receptor nuclear translocator-like; U2OS, human
osteosarcoma cell line; SAR, structure−activity relationship;
AcOH, acetic acid
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